Hi all. I'm a lab newbie and I need a little help. I completed three different Westerns and imaged all three but I found NO signal for any of my antibodies (three separate antibodies, incubated at room temp for 1hr; washings; secondary for an hour at room temp). I did a Ponceau stain and saw that the protein is there so I know that everything ran and transferred ok. I think I need to do my antibody incubation for a much longer time -- like overnight at 4 degree.

My question is: since I'm incubating with the exact same antibody, do I need to strip the blots and reblock and everything? Or can I just put the primary on and continue from there?